*2.6. RNA-seq Analysis*

Coverage analyses were performed to investigate the relationships between the identified 241 nt repeat regions in the *T. cruzi* strains as well as their closest upstream and downstream genes and respective range regions. The expression profile for the repeat region, upstream and downstream genes and the region between them were quantified based on the RNA-Seq data from *T. cruzi* strains (CL\_Brener Esmeraldo-Like, CL\_Brener Non-Esmeraldo-Like, and YC6) from NCBI, for the two different stages of the parasite (epimastigote and trypomastigote) with two biological replicates for *T. cruzi* CL\_Brener and three biological replicates for the other ones.

The accession numbers of SRAs from the NCBI of *T. cruzi* strains are as follows: CL\_Brener epimastigote (SRX1643253, SRX1643239) and trypomastigote (SRX1643235, SRX1643234); Y epimastigote (SRX574896, SRX574895, SRX574894) and trypomastigote (SRX574893, SRX574892, SRX574891, SRX574890).

To quantify the expression profile of these regions, we aligned the samples to the reference genome using Hisat2 version 2.1.0 [25] with "−k1" parameter, which allows only one alignment per read. Then, the raw counts were quantified based on the alignment of reads to each genome strain with the tool multiBamCov, and the coverage of the region was estimated with coverageBed from bedtools version 2.26.0 [26]. The EdgeR version 3.28.1 [27] was used to estimate the average expression profile between the replicates calculated and the log2 fold change between the stages trypomastigote and epimastigote for every strain and for every region.
